Identification of novel neutralizing determinants for protection against HCV.

BACKGROUND AND AIMS: HCV evasion of neutralizing antibodies (nAb) results in viral persistence and poses challenges to the development of an urgently needed vaccine. N-linked glycosylation of viral envelope proteins is a key mechanism for such evasion. To facilitate rational vaccine design, we aimed to identify determinants of protection of conserved neutralizing epitopes. APPROACH AND RESULTS: Using a reverse evolutionary approach, we passaged genotype 1a, 1b, 2a, 3a, and 4a HCV with envelope proteins (E1 and E2) derived from chronically infected patients without selective pressure by nAb in cell culture. Compared with the original viruses, HCV recombinants, engineered to harbor substitutions identified in polyclonal cell culture-passaged viruses, showed highly increased fitness and exposure of conserved neutralizing epitopes in antigenic regions 3 and 4, associated with protection from chronic infection. Further reverse genetic studies of acquired E1/E2 substitutions identified positions 418 and 532 in the N1 and N6 glycosylation motifs, localizing to adjacent E2 areas, as key regulators of changes of the E1/E2 conformational state, which governed viral sensitivity to nAb. These effects were independent of predicted glycan occupancy. CONCLUSIONS: We show how N-linked glycosylation motifs can trigger dramatic changes in HCV sensitivity to nAb, independent of glycan occupancy. These findings aid in the understanding of HCV nAb evasion and rational vaccine design, as they can be exploited to stabilize the structurally flexible envelope proteins in an open conformation, exposing important neutralizing epitopes. Finally, this work resulted in a panel of highly fit cell culture infectious HCV recombinants.

Exploring D-Lactate as a Biomarker for Acute Intestinal Necrosis in 2958 Patients: A Prospective Cross-Sectional Study.

BACKGROUND: Timely diagnosis of acute intestinal necrosis (AIN) is lifesaving, but challenging due to unclear clinical presentation. D-lactate has been proposed as an AIN biomarker. OBJECTIVES: We aimed to test the diagnostic performance in a clinical setting. METHODS: We performed a cross-sectional prospective study, including all adult patients with acute referral to a single tertiary gastrointestinal surgical department during 2015-2016 and supplemented by enrollment of high-risk in-hospital patients suspected of having AIN during 2016-2019. AIN was verified intraoperatively, and D-lactate was analyzed using an automatic spectrophotometric set-up. A D-lactate cut-off for AIN was estimated using the receiver operating characteristic curve. The performance according to patient subgroups was estimated using the area under the receiver operating characteristic curve (AUC). Given the exploratory nature of this study, a formal power calculation was not feasible. RESULTS: Forty-four AIN patients and 2914 controls were enrolled. The D-lactate cut-off was found to be 0.0925 mM. Due to lipemic interference, D-lactate could not be quantified in half of the patients, leaving 23 AIN patients and 1456 controls for analysis. The AUC for the diagnosis of AIN by D-lactate was 0.588 (95% confidence interval 0.475-0.712), with a sensitivity of 0.261 and specificity of 0.892. Analysis of high-risk patients showed similar results (AUC 0.579; 95% confidence interval 0.422-0.736). CONCLUSION: D-lactate showed low sensitivity for AIN in both average-risk and high-risk patients. Moreover, lipemic interference precluded valid spectrophotometric assessment of D-lactate in half of the patients, further disqualifying the clinical utility of D-lactate as a diagnostic marker for AIN.

Colorectal cancer detected by liquid biopsy 2 years prior to clinical diagnosis in the HUNT study.

BACKGROUND: Colorectal cancer (CRC) is often diagnosed in advanced stages. Circulating tumour DNA (ctDNA) has been proposed as an early diagnostic biomarker. However, as a screening tool, ctDNA has mainly been studied in selected populations at the time of clinical diagnosis. The aim of this study was to detect CRC by known ctDNA markers up to 2 years prior to clinical diagnosis. METHODS: In this case-control study, methylated ctDNA markers were detected in plasma samples from 106 healthy controls and 106 individuals diagnosed with CRC within 24 months following participation in The Trøndelag Health Study. RESULTS: The most specific single markers were BMP3, FLI1, IKZF1, SFRP1, SFRP2, NPTX2, SLC8A1 and VIM (specificity >70%). When combining these into a panel, the CRC sensitivity was 43% (95% CI 42.7-43.4) and the CRC specificity was 86% (95% CI 85.7-86.2). The findings were reproduced in an independent validation set of samples. CONCLUSIONS: Detection of known methylated ctDNA markers of CRC is possible up to 2 years prior to the clinical diagnosis in an unselected population resembling the screening setting. This study supports the hypothesis that some patients could be diagnosed earlier, if ctDNA detection was part of the CRC screening programme.

Promoter hypermethylation of SFRP1 as a prognostic and potentially predictive blood-based biomarker in patients with stage III or IV pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma remains one of the major causes of cancer-related mortality globally. Unfortunately, current prognostic biomarkers are limited, and no predictive biomarkers exist. This study examined promoter hypermethylation of secreted frizzled-related protein 1 (phSFRP1) in cfDNA as a prognostic biomarker and predictor of treatment effect in patients with metastatic FOLFIRINOX-treated PDAC and locally advanced PDAC. METHODS: We performed methylation-specific PCR of the SFRP1 genes' promoter region, based on bisulfite treatment. Survival was assessed as time-to-event data using the pseudo-observation method and analyzed with Kaplan-Meier curves and generalized linear regressions. RESULTS: The study included 52 patients with FOLFIRINOX-treated metastatic PDAC. Patients with unmethylated (um) SFRP1 (n = 29) had a longer median overall survival (15.7 months) than those with phSFRP1 (6.8 months). In crude regression, phSFRP1 was associated with an increased risk of death of 36.9% (95% CI 12.0%-61.7%) and 19.8% (95% CI 1.9-37.6) at 12 and 24-months, respectively. In supplementary regression analysis, interaction terms between SFRP1 methylation status and treatment were significant, indicating reduced benefit of chemotherapy. Forty-four patients with locally advanced PDAC were included. phSFRP1 was associated with an increased risk of death at 24-months CONCLUSIONS: This indicates that phSFRP1 is a clinically useful prognostic biomarker in metastatic PDAC and possibly in locally advanced PDAC. Together with existing literature, results could indicate the value of cfDNA-measured phSFRP1 as a predictive biomarker of standard palliative chemotherapy in patients with metastatic PDAC. This could facilitate personalized treatment of patients with metastatic PDAC.

The influence of Scandinavian presence on Greenlandic lactase persistence.

BACKGROUND: The study aims to estimate the prevalence of lactase non-persistence (LNP) among Greenlandic Inuit and Scandinavians living in Nuuk and East Greenland. The C to T transition in LCT - 13910 (rs4988235) is an autosomal inherited variant that provides the ability to lifelong lactase production, necessary to digest milk. The transition is very common in North European populations. However, LNP has only been sparsely studied in Greenland and never in Eastern Greenland, and genotype data has not previously been reported. METHODS: Whole blood samples were collected from 535 participants, and rs4988235 was typed using a PCR-based method. Ethnicity was defined by parents' place of birth. Results were compared between East and West Greenland and Inuit and Scandinavians using Pearson's Chi2 test. RESULTS: 82.2% of the participants were Inuit, and 17.8% were of Scandinavian ancestry. Among Inuit, 88.5% had LNP compared to 7.5% among Scandinavians (p < 0.001). The prevalence of LNP in Inuit varied significantly between East and West Greenland (p < 0.001). In the capital, 67.6% of Inuit had LNP compared to 98.6% in Tasiilaq and 100% in the villages around Tasiilaq. DISCUSSION: The difference in LNP between East and West Greenland and the Inuit and Scandinavian population found in our study suggests that the original Inuit population was lactose maldigesters. Our findings suggest that the -13910 T allele was introduced into the original Inuit population by the Danes.

Exploring I-FABP, endothelin-1 and L-lactate as biomarkers of acute intestinal necrosis: a case-control study.

OBJECTIVE: Acute intestinal necrosis (AIN) is a disease with devastating high mortality. AIN due to obstructed arterial blood flow has a blurred clinical presentation. Timely diagnosis is paramount, and a blood-based biomarker is warranted to increase patient survival. We aimed to assess intestinal fatty acid binding protein (I-FABP) and endothelin-1 as diagnostic biomarkers for AIN. To our knowledge, this is the first study exploring endothelin-1 in AIN patients from a general surgical population. DESIGN: We conducted a single-centre nested case-control study comparing acutely admitted AIN patients to age- and sex-matched non-AIN patients during 2015-2016. I-FABP and endothelin-1 were analysed using an enzyme-linked immunosorbent assay. L-lactate levels were also measured in all patients. Cut-offs were estimated using receiver operator characteristic curves, and the diagnostic performance was estimated using the area under the receiver operator characteristic curve (AUC). RESULTS: We identified 43 AIN patients and included 225 matched control patients. Median levels of I-FABP, endothelin-1 and L-lactate were 3550 (IQR: 1746-9235) pg/ml, 3.91 (IQR: 3.33-5.19) pg/ml and 0.92 (IQR: 0.74-1.45) mM in AIN patients and 1731 (IQR: 1124-2848) pg/ml, 2.94 (IQR: 2.32-3.82) pg/ml and 0.85 (IQR: 0.64-1.21) mM in control patients, respectively. The diagnostic performances of endothelin-1 and of I-FABP + endothelin-1 combined were moderate. Endothelin-1 alone revealed an AUC of 0.74 (0.67; 0.82). The sensitivity and specificity of endothelin-1 were 0.81 and 0.64, respectively. CONCLUSION: I-FABP and endothelin-1 are promising biomarkers for AIN, with moderate diagnostic performance compared with the commonly used biomarker L-lactate. PREREGISTRATION: ClinicalTrials.gov: NCT05665946.

Inactivated whole hepatitis C virus vaccine employing a licensed adjuvant elicits cross-genotype neutralizing antibodies in mice.

BACKGROUND & AIMS: A prophylactic vaccine is required to eliminate HCV as a global public health threat. We developed whole virus inactivated HCV vaccine candidates employing a licensed adjuvant. Further, we investigated the effects of HCV envelope protein modifications (to increase neutralization epitope exposure) on immunogenicity. METHODS: Whole virus vaccine antigen was produced in Huh7.5 hepatoma cells, processed using a multistep protocol and formulated with adjuvant (MF-59 analogue AddaVax or aluminium hydroxide). We investigated the capacity of IgG purified from the serum of immunized BALB/c mice to neutralize genotype 1-6 HCV (by virus neutralization assays) and to bind homologous envelope proteins (by ELISA). Viruses used for immunizations were (i) HCV5aHi with strain SA13 envelope proteins and modification of an O-linked glycosylation site in E2 (T385P), (ii) HCV5aHi(T385) with reversion of T385P to T385, featuring the original E2 sequence determined in vivo and (iii) HCV5aHi(ΔHVR1) with deletion of HVR1. For these viruses, epitope exposure was investigated using human monoclonal (AR3A and AR4A) and polyclonal (C211 and H06) antibodies in neutralization assays. RESULTS: Processed HCV5aHi formulated with AddaVax induced antibodies that efficiently bound homologous envelope proteins and broadly neutralized cultured genotype 1-6 HCV, with half maximal inhibitory concentrations of between 14 and 192 µg/ml (mean of 36 µg/ml against the homologous virus). Vaccination with aluminium hydroxide was less immunogenic. Compared to HCV5aHi(T385) with the original E2 sequence, HCV5aHi with a modified glycosylation site and HCV5aHi(ΔHVR1) without HVR1 showed increased neutralization epitope exposure but similar immunogenicity. CONCLUSION: Using an adjuvant suitable for human use, we developed inactivated whole HCV vaccine candidates that induced broadly neutralizing antibodies, which warrant investigation in further pre-clinical studies. LAY SUMMARY: A vaccine against hepatitis C virus (HCV) is needed to prevent the estimated 2 million new infections and 400,000 deaths caused by this virus each year. We developed inactivated whole HCV vaccine candidates using adjuvants licensed for human use, which, following immunization of mice, induced antibodies that efficiently neutralized all HCV genotypes with recognized epidemiological importance. HCV variants with modified envelope proteins exhibited similar immunogenicity as the virus with the original envelope proteins.

Mortality in Greenlanders with chronic hepatitis B virus infection.

In-depth reviewing of all medical records and clinical databases concluded a 7-year shorter lifespan among Greenlanders infected with hepatitis B virus (HBV) compared with non-infected. Mortality did not associate with liver disease or any other specific disease entity. A possible mechanism for the reduced lifespan is subclinical inflammation that may be augmented by chronic viral infection. We hypothesized that chronic HBV infection contributes to this process causing a reduced life span. We added measurement of two markers of inflammation to the 10-year follow-up on our study of HBV among 50- through 69-years-old subjects in Greenland. The markers were YKL40 related to liver disease and hsCRP as a global marker of inflammation. Survival was evaluated using Cox regression with time until death entered as dependent variable and age, sex, smoking, alcohol intake, BMI, the presence of HBsAg and one marker of inflammation as explanatory variables. Forty-eight percent of participants with chronic HBV infection were alive after 10 years compared with 65% of participants without infection (p = 0.003). Survival associated with age (p < 0.001), BMI (p = 0.003) and both YKL40 and hsCRP (both, p < 0.001). Harbouring HBV influenced 10-year survival in the Cox regression after adjusting for age, sex, BMI, smoking, alcohol intake and inflammation. In conclusion, chronic low-grade inflammation and being infected with HBV were independent markers of mortality in otherwise healthy subjects. Thus, the 7-year shorter lifespan among Greenlanders with chronic HBV infection seems related to the long-lasting infection. Our findings call for caution in perceiving a chronic infection as benign.

Mortality and cause of death in persons with chronic hepatitis B virus infection versus healthy persons from the general population in Denmark.

The study aimed to determine adjusted all-cause mortality and cause of death in persons with chronic hepatitis B virus (HBV) infection compared with age- and sex-matched persons from the general population. We used nationwide registers to identify persons aged ≥18 years with chronic HBV infection in 2002-2017 in Denmark and included 10 age- and sex-matched controls for each. Follow-up was from 6 months after diagnosis until death, emigration, or 31 December 2017. Mortality rate ratios (MRRs) adjusted for age, sex, employment, origin and comorbidity were calculated using Poisson regression. Unadjusted cause-specific mortality rate ratios with 95% confidence intervals were calculated assuming a Poisson distribution. A total of 6988 persons with chronic HBV infection and 69,847 controls were included. During a median follow-up of 7.7 years (range 0.0-15.5), 315 (5%) persons with-and 1525 (2%) without-chronic HBV infection died. The adjusted all-cause MRR was 1.5 (95% CI 1.2-2.0). Persons with chronic HBV infection had increased mortality due to liver disease including hepatocellular carcinoma (MRR 12.3 [8.6-17.7]), external causes (MRR 3.3 [2.5-4.7]), endocrine disease (MRR 3.2 [1.8-5.4]), genitourinary disease (MRR 3.2 [1.2-7.6]) and neoplasms (except hepatocellular carcinoma; MRR 1.6 [1.2-2.0]). In conclusion, this study showed an increased all-cause mortality in persons with chronic HBV infection in comparison with age- and sex-matched persons without chronic HBV infection which remained after adjustment for several confounding factors. Excess mortality was mainly associated with liver disease, but also external factors, endocrine disease, genitourinary disease and neoplasms (excluding hepatocellular carcinoma).

Early Laboratory Diagnosis of COVID-19 by Antigen Detection in Blood Samples of the SARS-CoV-2 Nucleocapsid Protein.

The purpose of this study was to characterize the diagnostic performance of a newly developed enzyme-linked immunosorbent assay (ELISA) for detection of SARS-CoV-2 nucleocapsid protein (NP) in blood. Blood samples were collected during hospitalization of 165 inpatients with PCR-confirmed SARS-CoV-2 infection and from 505 outpatients predominantly with relevant symptoms of COVID-19 simultaneously with PCR testing. For the 143 inpatients who had their first blood sample collected within 2 weeks after PCR-confirmed infection, the diagnostic sensitivity of the ELISA was 91.6%. The mean NP concentration of the 131 ELISA-positive blood samples was 1,734 pg/ml (range, 10 to 3,840 pg/ml). An exponential decline in NP concentration was observed for 368 blood samples collected over the first 4 weeks after PCR-confirmed SARS-CoV-2 infection, and all blood samples taken later had an NP concentration below the 10-pg/ml diagnostic cutoff. The diagnostic sensitivity of the ELISA was 81.4% for the 43 blood samples collected from outpatients with a simultaneous positive PCR test, and the mean NP concentration of the 35 ELISA-positive samples was 157 pg/ml (range, 10 to 1,377 pg/ml). For the 462 outpatients with a simultaneous negative PCR test, the diagnostic specificity of the ELISA was 99.8%. In conclusion, the SARS-CoV-2 NP ELISA is a suitable laboratory diagnostic test for COVID-19, particularly for hospitals, where blood samples are readily available and screening of serum or plasma by ELISA can facilitate prevention of nosocomial infections and reduce the requirement for laborious swab sampling and subsequent PCR analysis to confirmatory tests only.

Global evolutionary analysis of chronic hepatitis C patients revealed significant effect of baseline viral resistance, including novel non-target sites, for DAA-based treatment and retreatment outcome.

Direct-acting antivirals (DAAs) have proven highly effective against chronic hepatitis C virus (HCV) infection. However, some patients experience treatment failure, associated with resistance-associated substitutions (RASs). Our aim was to investigate the complete viral coding sequence in hepatitis C patients treated with DAAs to identify RASs and the effects of treatment on the viral population. We selected 22 HCV patients with sustained virologic response (SVR) to match 21 treatment-failure patients in relation to HCV genotype, DAA regimen, liver cirrhosis and previous treatment experience. Viral-titre data were compared between the two patient groups, and HCV full-length open reading frame deep-sequencing was performed. The proportion of HCV NS5A-RASs at baseline was higher in treatment-failure (82%) than matched SVR patients (25%) (p = .0063). Also, treatment failure was associated with slower declines in viraemia titres. Viral population diversity did not differ at baseline between SVR and treatment-failure patients, but failure was associated with decreased diversity probably caused by selection for RAS. The NS5B-substitution 150V was associated with sofosbuvir treatment failure in genotype 3a. Further, mutations identified in NS2, NS3-helicase and NS5A-domain-III were associated with DAA treatment failure in genotype 1a patients. Six retreated HCV patients (35%) experienced 2nd treatment failure; RASs were present in 67% compared to 11% with SVR. In conclusion, baseline RASs to NS5A inhibitors, but not virus population diversity, and lower viral titre decline predicted HCV treatment failure. Mutations outside of the DAA targets can be associated with DAA treatment failure. Successful DAA retreatment in patients with treatment failure was hampered by previously selected RASs.

Metabolic fingerprint of progression of chronic hepatitis B: changes in the metabolome and novel diagnostic possibilities.

INTRODUCTION: Chronic hepatitis B (CHB) affects 257 million individuals worldwide with an annual estimated mortality rate of 880,000 individuals. Accurate diagnosis of the stage of disease is difficult, and there is considerable uncertainty concerning the optimal point in time, when treatment should be started. OBJECTIVES: By analyzing and comparing the metabolomes of patients at different stages of CHB and comparing them to healthy individuals, we want to determine the metabolic signature of disease progression and develop a more accurate metabolome-based method for diagnosis of disease progression ultimately giving a better basis for treatment decisions. METHODS: In this study, we used the combination of transient elastography and serum metabolomics of 307 serum samples from a group of 90 patients with CHB before and under treatment (with a follow-up time up to 10 years) at different progression stages over the clinical phases and 43 healthy controls.. RESULTS: Our data show that the metabolomics approach can successfully discover CHB changing from the immune tolerance to the immune clearance phase and show distinctive metabolomes from different medical treatment stages. Perturbations in ammonia detoxification, glutamine and glutamate metabolism, methionine metabolism, dysregulation of branched-chain amino acids, and the tricarboxylic acid (TCA) cycle are the main factors involved in the progression of the disease. Fluctuations increasing in aspartate, glutamate, glutamine, methionine and 13 other metabolites are fingerprints of progression. CONCLUSIONS: The metabolomics approach may expand the diagnostic armamentarium for patients with CHB. This method can provide a more detailed decision basis for starting medical treatment.

Cell-free DNA promoter hypermethylation as a diagnostic marker for pancreatic ductal adenocarcinoma - An external validation study.

BACKGROUND: We recently identified a diagnostic prediction model based on promoter hypermethylation of eight selected genes in plasma cell-free (cf) DNA, which showed promising results as a diagnostic biomarker for pancreatic ductal adenocarcinoma (PDAC). The aim of the present study was to validate this biomarker profile in an external patient cohort and examine any additional effect of serum CA 19-9. METHODS: Patients with PDAC (n = 346, stage I-IV) and chronic pancreatitis (n = 25) were included. Methylation-specific PCR of a 28-gene panel was performed on serum cfDNA samples. The previously developed diagnostic prediction model (age>65 years, BMP3, RASSF1A, BNC1, MESTv2, TFPI2, APC, SFRP1 and SFRP2) was validated alone and in combination with serum CA 19-9 in this external patient cohort. RESULTS: Patients with PDAC had a higher number of hypermethylated genes (mean 8.11, 95% CI 7.70-8.52) than patients with chronic pancreatitis (mean 5.60, 95% CI 4.42-6.78, p = 0.011). Validation of the diagnostic prediction model yielded an AUC of 0.77 (95% CI 0.69-0.84). The combination of serum CA 19-9 and our test had an AUC of 0.93 (95% CI 0.89-0.96) in the primary study and 0.85 (95% CI 0.79-0.91) in the validation study. CONCLUSION: In this validation study, PDAC was associated with a higher number of hypermethylated genes in serum cfDNA than chronic pancreatitis. Our diagnostic test was superior to the predictive value of serum CA 19-9 alone in both the primary and the validation study. The combination of our test with CA 19-9 may serve as a clinically useful diagnostic biomarker for PDAC.

Chronic Postoperative Pain After Hysterectomy for Endometrial Cancer: A Metabolic Profiling Study.

INTRODUCTION: One out of seven women will develop a state of chronic postoperative pain following robot-assisted hysterectomy for endometrial cancer. Recently, metabolic studies have indicated that circulating lipids and lipoproteins could act as nociceptive modulators and thereby influence the induction and perpetuation of pain. The objectives of this explorative study were (1) to examine the preoperative serologic variations in concentrations of lipids, lipoproteins, and various low-molecular metabolites in patients with and without chronic postoperative pain after robot-assisted hysterectomy and (2) to explore if any of these serological biomarkers were predictive for development of chronic postoperative pain. MATERIALS AND METHODS: The study was designed as a nested case-control study within a cohort of women treated for endometrial cancer with robot-assisted laparoscopic hysterectomy. Twenty-six women with chronic postoperative pain were matched on age and body mass index with fifty-two controls without chronic postoperative pain, and metabolic profiling of preoperatively drawn blood samples from a biobank was performed by means of nuclear magnetic resonance spectroscopy. RESULTS: Nineteen metabolites, including cholesterol, cholesteryl ester, linoleic acid, phospholipids, lipids, and triglycerides had statistically significant higher concentrations in a subgroup of patients who would develop chronic postoperative pain on a later stage compared to the group of patients who would not develop chronic postoperative pain (p < 0.05). A sparse Partial Least Squares-Discriminant Analysis model explained 38.1% of the variance and had a predictive accuracy of 73.1%. CONCLUSIONS: This explorative study substantiates the hypothesis that certain lipids, lipoproteins, and fatty acids are associated with chronic postoperative pain.

Consequences of parenteral iron-dextran loading investigated in minipigs. A new model of transfusional iron overload.

Patients with blood transfusion-dependent anemias develop transfusional iron overload (TIO), which may cause cardiosiderosis. In patients with an ineffective erythropoiesis, such as thalassemia major, common transfusion regimes aim at suppression of erythropoiesis and of enteral iron loading. Recent data suggest that maintaining residual, ineffective erythropoiesis may protect from cardiosiderosis. We investigated the common consequences of TIO, including cardiosiderosis, in a minipig model of iron overload with normal erythropoiesis. TIO was mimicked by long-term, weekly iron-dextran injections. Iron-dextran loading for around one year induced very high liver iron concentrations, but extrahepatic iron loading, and iron-induced toxicities were mild and did not include fibrosis. Iron deposits were primarily in reticuloendothelial cells, and parenchymal cardiac iron loading was mild. Compared to non-thalassemic patients with TIO, comparable cardiosiderosis in minipigs required about 4-fold greater body iron loads. It is suggested that this resistance against extrahepatic iron loading and toxicity in minipigs may at least in part be explained by a protective effect of the normal erythropoiesis, and additionally by a larger total iron storage capacity of RES than in patients with TIO. Parenteral iron-dextran loading of minipigs is a promising and feasible large-animal model of iron overload, that may mimic TIO in non-thalassemic patients.

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants.

Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance-associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1-4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156-RASs were not maintained. For genotypes 1 and 2, persistence of 156-RASs depended on genome-wide substitution networks, co-selected under continued PI treatment and identified by next-generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156-RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre-existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156-RASs, we observed genome-wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1-6 revealed 156-RASs as key determinants of high-level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1-3 156-variants, which might pose a threat to clinically relevant combination treatments.

Hepatitis B and C in the adult population of Bissau, Guinea-Bissau: a cross-sectional survey.

OBJECTIVE: Hepatitis B virus (HBV) and hepatitis C virus (HCV) are prevalent in West Africa. To address the WHO 2030 goals of a 90% reduction in incidence and a 65% reduction in mortality for both infections, we assessed the prevalence of HBV and HCV from surveys in the general population. METHODS: Participants in this cross-sectional survey were included from randomly selected houses in a demographic surveillance site in Bissau, Guinea-Bissau. Participants were interviewed and had a blood sample drawn for viral analyses (HBsAg, anti-HBs, anti-HBc, anti-HCV and HCV RNA). Risk factors of HBV and HCV infection were determined by binomial regression adjusted for sex and age. RESULTS: A total of 2715 participants were included in this study. The overall HBsAg prevalence was 18.7% (95% CI: 17.3-20.2%). HBsAg was associated with male sex (adjusted risk ratio (aRR): 1.64), and prevalence decreased with age >34 years. HBV exposure was found in 91.9% of participants. Although 72.6% of individuals without sexual debut had been exposed to HBV, ever engaging in a sexual relationship was associated with higher risk of HBV exposure (aRR 1.18). The anti-HCV prevalence was 0.5% (95% CI: 0.3-0.9%), and 78.6% of those had detectable HCV RNA. Risk factors for anti-HCV sero-positivity were age above 55 (aRR 10.60), a history of blood transfusion (aRR 5.07) and being in a polygamous marriage (aRR 3.52). CONCLUSION: In Guinea-Bissau initiatives to implement treatment and widespread testing are needed to reach the WHO 2030 goals.

OBJECTIF: Le virus de l'hépatite B (VHB) et le virus de l'hépatite C (VHC) sont répandus en Afrique de l'Ouest. Pour atteindre les objectifs de 2030 de l'OMS d'une réduction de 90% de l'incidence et de 65% de la mortalité pour les deux infections, nous avons évalué la prévalence du VHB et du VHC à partir d'enquêtes dans la population générale. MÉTHODES: Les participants inclus dans cette enquête transversale provenaient de foyers sélectionnés au hasard dans un site de surveillance démographique à Bissau, en Guinée-Bissau. Les participants ont été interrogés et ont subi un prélèvement d'échantillon de sang pour des analyses virales (HBsAg, anti-HBs, anti-HBc, anti-HCV et ARN du HCV). Les facteurs de risque d'infection par le VHB et le VHC ont été déterminés par la régression binomiale ajustée en fonction du sexe et de l'âge. RÉSULTATS: 2.715 participants ont été inclus dans cette étude. La prévalence globale de l'HBsAg était de 18,7% (IC95%: 17,3-20,2%). L'HBsAg était associé au sexe masculin (rapport de risque ajusté (aRR): 1,64), et la prévalence diminuait avec l'âge >34 ans. Une exposition au VHB a été observée chez 91,9% des participants. Bien que 72,6% des personnes sans début d'activité sexuelle aient été exposées au VHB, le fait de s'engager dans des relations sexuelles était associé à un risque plus élevé d'exposition au VHB (aRR: 1,18). La prévalence d'anti-VHC était de 0,5% (IC95%: 0,3-0,9%) et 78,6% d'entre eux avaient de l'ARN du VHC détectable. Les facteurs de risque de séropositivité anti-VHC étaient l'âge de plus de 55 ans (aRR: 10,60), les antécédents de transfusion sanguine (aRR: 5,07) et le fait d'être dans un mariage polygame (aRR: 3,52). CONCLUSION: En Guinée-Bissau, des initiatives pour mettre en œuvre un traitement et des tests généralisés sont nécessaires pour atteindre les objectifs de l'OMS 2030.

Use of genetic testing for hypolactasia trait in the North Denmark Region.

OBJECTIVE: Lactose intolerance (LI) may be considered in patients with unspecific gastrointestinal symptoms, but there is no clear consensus on when and how to diagnose the disorder. The LCT-13910 CC genotype is associated with acquired primary lactase deficiency (adult-type hypolactasia; ATH). We aimed to describe the number of tests and test results in the North Denmark Region considering patient age, geographical origin and repeated testing. METHODS: Retrospective evaluation of the polymerase chain reaction-based LCT-13910 genotype tests registered in the clinical laboratory information system (LABKA II) with data linkage to Danish nationwide registers. RESULTS: Between 18 May 2007 and 31 December 2018, a total of 23,560 individuals were tested. There was a sevenfold increase in the number of tests performed during the study period. About 9.8% of the tests performed in 2018 were repeated testing in the same individuals. Overall, 8.8% of tested individuals were younger than 5 years, 90.7% were of Danish origin and 5.5% originated from outside of Europe. The LCT-13910 CC genotype was identified in 13.3% of all tested individuals, in 16.0% of children younger than 5 years, in 6.8% of Danish individuals and in 90.9% originating from outside of Europe. CONCLUSIONS: In the North Denmark Region, a marked increase in the use of genetic testing for hypolactasia was observed and repeated testing was frequent. Furthermore, the use of the test and the test results were dependent on patient age and geographical origin. Results inform the debate on when and how to use genetic testing in the diagnosing of LI.

Implementation of treatment recommendations for chronic hepatitis B in patients attending specialized hospital care in Denmark - a region wide study.

Objective: To evaluate implementation of national guideline recommendations on treatment initiation for chronic hepatitis B (CHB) in Denmark.Methods: Using DANHEP, a nationwide cohort of chronic hepatitis B and C patients attending specialized hospital care in Denmark, we performed a descriptive cohort study from January 2002 through December 2017. We identified patients with CHB in 3 of 5 Danish regions, with at least two hospital/outpatient clinic visits during the study period.Results: We identified 990 CHB patients who remained untreated throughout the study period, and 265 who initiated treatment. At their last visit 952/990 (96%, 95% CI 95-97) untreated patients did not meet current national criteria for treatment initiation while 198/265 (75%, 95% CI 69-80) who initiated treatment met the national criteria. Overall, 198/236 (84%, 95% CI 79-88) who met national treatment criteria, initiated treatment.Conclusion: The majority of CHB patients received care in line with national guideline recommendations for treatment initiation. We found that only few patients eligible for treatment remained untreated. However, a fourth of patients who received treatment were not eligible according to national guidelines.

Differential effect of surgical manipulation on gene expression in normal breast tissue and breast tumor tissue.

BACKGROUND: Gene expression profiles of normal and tumor tissue reflect both differences in biological processes taking place in vivo and differences in response to stress during surgery and sample handling. The effect of cold (room temperature) ischemia in the time interval between surgical removal of the specimen and freezing is described in a few studies. However, not much is known about the effect of warm (body temperature) ischemia during surgery. METHODS: Three women with primary operable breast cancer underwent in situ biopsies from normal breast and tumor tissue prior to radical mastectomy. Ex vivo biopsies from normal and tumor tissue were collected immediately after surgical excision. The putative effects on gene expression of malignancy (tumor versus normal), surgical manipulation (post- versus pre-surgical) and interaction between the two (differences in effect of surgical manipulation on tumor and normal samples) were investigated simultaneously by Generalized Estimating Equation (GEE) analysis in this self-matched study. RESULTS: Gene set enrichment analysis (GSEA) demonstrates a marked difference in effect of surgical manipulation on tumor compared to normal tissue. Interestingly, a large proportion of pathways affected by ischemia especially in tumor tissue are pathways considered to be specifically up regulated in tumor tissue compared to normal. CONCLUSION: The results of this study suggest that a large contribution to this differential expression originates from altered response to stress in tumor cells rather than merely representing in vivo differences. It is important to bear this in mind when using gene-expression analysis to deduce biological function, and when collecting material for gene expression profiling.